WO2011072152A1 - Mass spectrometry of steroidal compounds in multiplex samples - Google Patents

Mass spectrometry of steroidal compounds in multiplex samples Download PDF

Info

Publication number
WO2011072152A1
WO2011072152A1 PCT/US2010/059746 US2010059746W WO2011072152A1 WO 2011072152 A1 WO2011072152 A1 WO 2011072152A1 US 2010059746 W US2010059746 W US 2010059746W WO 2011072152 A1 WO2011072152 A1 WO 2011072152A1
Authority
WO
WIPO (PCT)
Prior art keywords
vitamin
sample
ptad
samples
ions
Prior art date
Application number
PCT/US2010/059746
Other languages
English (en)
French (fr)
Inventor
Brett Holmquist
Nigel J. Clarke
Original Assignee
Quest Diagnostics Investments Incorporated
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Quest Diagnostics Investments Incorporated filed Critical Quest Diagnostics Investments Incorporated
Priority to EP19182389.7A priority Critical patent/EP3693734A3/en
Priority to CN201080063451.XA priority patent/CN102812356B/zh
Priority to CA2783708A priority patent/CA2783708C/en
Priority to US13/514,892 priority patent/US9034653B2/en
Priority to JP2012543287A priority patent/JP5739903B2/ja
Priority to EP10836702.0A priority patent/EP2510347B1/en
Publication of WO2011072152A1 publication Critical patent/WO2011072152A1/en
Priority to US14/715,153 priority patent/US9506937B2/en
Priority to US15/362,210 priority patent/US10955424B2/en
Priority to US17/208,564 priority patent/US11808773B2/en
Priority to US18/364,356 priority patent/US20230375575A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0027—Methods for using particle spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/21—Hydrocarbon
    • Y10T436/212—Aromatic

Definitions

  • the invention relates to the quantitative measurement of steroidal compounds by mass spectrometry.
  • the invention relates to methods for quantitative measurement of steroidal compounds from multiple samples by mass spectrometry.
  • Steroidal compounds are any of numerous naturally occurring or synthetic fat-soluble organic compounds having as a basis 17 carbon atoms arranged in four rings and including the sterols and bile acids, adrenal and sex hormones, certain natural drugs such as digitalis compounds, as well as certain vitamins and related compounds (such as vitamin D, vitamin D analogues, and vitamin D metabolites).
  • vitamin D is an anthyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • Vitamin D can be made de novo in the skin by exposure to sunlight or it can be absorbed from the diet.
  • vitamin D 2 ergocalciferol
  • vitamin D 3 cholecalciferol
  • Vitamin D 3 is the form synthesized de novo by animals. It is also a common supplement added to milk products and certain food products produced in the United States. Both dietary and intrinsically synthesized vitamin D 3 must undergo metabolic activation to generate the bioactive metabolites.
  • the initial step of vitamin D 3 activation occurs primarily in the liver and involves hydroxylation to form the intermediate metabolite 25- hydroxycholecalciferol (calcifediol; 250HD 3 ).
  • Calcifediol is the major form of Vitamin D 3 in circulation. Circulating 250HD 3 is then converted by the kidney to form 1 ,25-dihydroxyvitamin D 3 (calcitriol; l,25(OH) 2 D 3 ), which is generally believed to be the metabolite of Vitamin D 3 with the highest biological activity.
  • Vitamin D 2 is derived from fungal and plant sources. Many over-the-counter dietary supplements contain ergocalciferol (vitamin D 2 ) rather than cholecalciferol (vitamin D 3 ).
  • Drisdol the only high-potency prescription form of vitamin D available in the United States, is formulated with ergocalciferol.
  • Vitamin D 2 undergoes a similar pathway of metabolic activation in humans as Vitamin D3, forming the metabolites 250HD 2 and l,25(OH) 2 D 2 .
  • Vitamin D 2 and vitamin D3 have long been assumed to be biologically equivalent in humans, however recent reports suggest that there may be differences in the bioactivity and bioavailability of these two forms of vitamin D (Armas et. al, (2004) J. Clin. Endocrinol. Metab. 89:5387-5391).
  • vitamin D the inactive vitamin D precursor
  • serum levels of 25-hydroxyvitamin D3, 25-hydroxyvitamin D 2 , and total 25- hydroxyvitamin D are useful indices of vitamin D nutritional status and the efficacy of certain vitamin D analogs.
  • the measurement of 250HD is commonly used in the diagnosis and management of disorders of calcium metabolism. In this respect, low levels of 250HD are indicative of vitamin D deficiency associated with diseases such as hypocalcemia,
  • hypophosphatemia secondary hyperparathyroidism, elevated alkaline phosphatase, osteomalacia in adults and rickets in children.
  • elevated levels of 250HD distinguishes this disorder from other disorders that cause hypercalcemia.
  • Measurement of l,25(OH) 2 D is also used in clinical settings. Certain disease states can be reflected by circulating levels of l,25(OH) 2 D, for example kidney disease and kidney failure often result in low levels of 1,25(0 H) 2 D. Elevated levels of l,25(OH) 2 D may be indicative of excess parathyroid hormone or can be indicative of certain diseases such as sarcoidosis or certain types of lymphomas.
  • the present invention provides methods for detecting the amount of a steroidal compound in each of a plurality of test samples with a single mass spectrometric assay.
  • the methods include processing each test sample differently to form a plurality of processed samples, wherein as a result of the processing, the steroidal compound in each processed sample is distinguishable by mass spectrometry from the steroidal compound in other processed samples; combining the processed samples to form a multiplex sample; subjecting the multiplex sample to an ionization source under conditions suitable to generate one or more ions detectable by mass spectrometry, wherein one or more ions generated from the steroidal compound from each processed sample are distinct from one or more ions from the steroidal compound from the other processed samples; detecting the amount of one or more ions from the steroidal compound from each processed sample by mass spectrometry; and relating the amount of one or more ions from the steroidal compound from each processed sample to the amount of the steroidal compound in each test sample.
  • processing a test sample comprises subjecting each test sample to a different derivatizing agent under conditions suitable to generate derivatized steroidal compounds.
  • one test sample may be processed without subjecting the sample to a derivatizing agent.
  • the different derivatizing agents used in the processing of the plurality of test samples are isotopic variants of each another.
  • the different derivatizing agents are Cookson-type derivatizing agents; such as Cookson-type derivatization agents selected from the group consisting of 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-l,2,4-triazoline-3,5- dione (DMEQTAD), 4-(4-nitrophenyl)-l,2,4-triazoline-3,5- dione (NPTAD), 4- ferrocenylmethyl-l,2,4-triazoline-3,5-dione (FMTAD), and isotopic variants thereof.
  • PTAD 4-phenyl-l,2,4-triazoline-3,5-dione
  • DEQTAD 4-[2-(6,7-d
  • the Cookson-type derivatizing agents are isotopic variants of 4-phenyl- l,2,4-triazoline-3,5-dione (PTAD).
  • the plurality of samples comprises two samples, a first Cookson-type derivatizing reagent is 4-phenyl-l,2,4-triazoline-3,5-dione
  • Cookson-type derivatizing reagent is C6-4-phenyl-l,2,4-triazoline-3,5- dione ( 13 C 6 -PTAD).
  • the steroidal compound is a vitamin D or vitamin D related compound.
  • the steroidal compound is selected from the group consisting of vitamin D 2 , vitamin D 3 , 25-hydroxyvitamin D 2 (250HD 2 ), 25-hydroxyvitamin D 3 (250HD 3 ), l ,25-dihydroxyvitamin D 2 (la,250HD 2 ), and l ,25-dihydroxyvitamin D 3
  • the steroidal compound is 25-hydroxyvitamin D 2 (250HD 2 ) or 25-hydroxyvitamin D 3 (250HD 3 ).
  • the methods described above may be conducted for the analysis of two or more steroidal compounds in each of a plurality of test samples.
  • the two or more steroidal compounds in each test sample may include at least one steroidal compound selected from the group consisting of 25-hydroxyvitamin D 2 (250HD 2 ) and 25- hydroxyvitamin D 3 (250HD 3 ).
  • the two or more steroidal compounds in each test sample are 25-hydroxyvitamin D 2 (250HD 2 ) and 25-hydroxyvitamin D 3 (250HD 3 ).
  • the amount of one or more vitamin D or vitamin D related compounds in each of two test samples is determined with a single mass spectrometric assay.
  • a first processed sample is generated by subjecting a first test sample to a first isotopic variant of 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) under conditions suitable to generate one or more vitamin D or vitamin D related derivatives;
  • a second processed sample is generated by subjecting a second test sample to a second isotopic variant of 4-phenyl- 1,2,4- triazoline-3,5-dione (PTAD) under conditions suitable to generate one or more vitamin D or vitamin D related derivatives, wherein the first and second isotopic variant of PTAD are distinguishable by mass spectrometry;
  • the first processed sample is mixed with the second processed sample to form a multiplex sample; one or more vitamin D or vitamin D related derivatives from each processed sample in the multiplex sample are subjected to an ionization source under conditions suitable to generate one
  • the first isotopic variant of 4-phenyl-l,2,4-triazoline- 3,5-dione is 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD)
  • the second isotopic variant of 4-phenyl-l,2,4-triazoline- 3,5-dione is 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD)
  • PTAD 4-phenyl-l,2,4-triazoline-3,5-dione
  • the one or more vitamin D or vitamin D related compounds are selected from the group consisting of 25-hydroxyvitamin D 2 (250HD 2 ) and 25- hydroxyvitamin D 3 (250HD 3 ). In some related specific embodiments, the one or more vitamin D or vitamin D related compounds include 25-hydroxyvitamin D 2 (250HD 2 ) and 25- hydroxyvitamin D 3 (250HD 3 ). In some related specific embodiments, the one or more vitamin D or vitamin D related compounds are 25-hydroxyvitamin D 2 (250HD 2 ) and 25-hydroxyvitamin D 3 (250HD 3 ).
  • the multiplex sample is subjected to an extraction column and an analytical column prior to being subjected to an ionization source.
  • the extraction column is a solid-phase extraction (SPE) column.
  • the extraction column is a turbulent flow liquid chromatography (TFLC) column.
  • the analytical column is a high performance liquid chromatography (HPLC) column.
  • mass spectrometry may be tandem mass spectrometry.
  • tandem mass spectrometry may be conducted by any method known in the art, including for example, multiple reaction monitoring, precursor ion scanning, or product ion scanning.
  • steroidal compounds may be ionized by any suitable ionization technique known in the art.
  • the ionization source is a laser diode thermal desorption (LDTD) ionization source.
  • LDTD laser diode thermal desorption
  • test samples comprise biological samples, such as plasma or serum.
  • multiplex sample refers to a sample prepared by pooling two or more samples to form the single "multiplex" sample which is then subject to mass spectrometric analysis.
  • two or more test samples are each processed differently to generate multiple differently processed samples. These multiple differently processed samples are then pooled to generate a single "multiplex" sample, which is then subject to mass spectrometric analysis.
  • steroidal compound refers to any of numerous naturally occurring or synthetic fat- soluble organic compounds having as a basis 17 carbon atoms arranged in four rings and including the sterols and bile acids, adrenal and sex hormones, certain natural drugs such as digitalis compounds, as well as certain vitamins and related compounds (such as vitamin D, vitamin D analogues, and vitamin D metabolites).
  • vitamin D or vitamin D related compound refers to any natural or synthetic form of vitamin D, or any chemical species related to vitamin D generated by a transformation of vitamin D, such as intermediates and products of vitamin D metabolism.
  • vitamin D may refer to one or more of vitamin D 2 and vitamin D y Vitamin D may also be referred to as "nutritional" vitamin D to distinguish from chemical species generated by a transformation of vitamin D.
  • Vitamin D related compounds may include chemical species generated by biotransformation of analogs of, or a chemical species related to, vitamin D 2 or vitamin D y Vitamin D related compounds, specifically vitamin D metabolites, may be found in the circulation of an animal and/or may be generated by a biological organism, such as an animal.
  • Vitamin D metabolites may be metabolites of naturally occurring forms of vitamin D or may be metabolites of synthetic vitamin D analogs.
  • vitamin D related compounds may include one or more vitamin D metabolites selected from the group consisting of 25-hydroxyvitamin D 3 , 25-hydroxyvitamin D 2 , l ,25-dihydroxyvitamin D 3 and la,25- dihydroxyvitamin D r
  • a derivatizing agent is an agent that is reacted with another substance to derivatize the substance.
  • PTAD 4-phenyl- l,2,4-triazoline-3,5-dione
  • PTAD is a derivatizing reagent that may be reacted with a vitamin D metabolite to form a PTAD-derivatized vitamin D metabolite.
  • differentiate derivatizing agents are derivatizing agents that are distinguishable by mass spectrometry.
  • two isotopic variants of the same derivatizing agent are distinguishable by mass spectrometry.
  • halogenated variants of the same derivatizing agent are also distinguishable by mass spectrometry.
  • halogenated and non-halogenated versions of the same Cookson-type agent such as 4- phenyl-l,2,4-triazoline-3,5-dione (PTAD)
  • PTAD 4- phenyl-l,2,4-triazoline-3,5-dione
  • two halogenated versions of the same Cookson-type agent but halogenated with different halogens or with different numbers of halogens, may be used.
  • Cookson-type agents such as 4- phenyl-l,2,4-triazoline-3,5-dione (PTAD), 4-methyl-l,2,4-triazoline-3,5-dione (MTAD), and 4- (4-nitrophenyl)-l,2,4-triazoline-3,5- dione (NPTAD), may be used.
  • PTAD 4- phenyl-l,2,4-triazoline-3,5-dione
  • MTAD 4-methyl-l,2,4-triazoline-3,5-dione
  • NPTAD 4- (4-nitrophenyl)-l,2,4-triazoline-3,5- dione
  • the names of derivatized forms of steroidal compounds include an indication as to the nature of derivatization.
  • the PTAD derivative of 25- hydroxyvitamin D 2 is indicated as PTAD-25-hydroxyvitamin D 2 (or PTAD-250HD 2 ).
  • a "Cookson-type derivatizing agent” is a 4-substituted 1,2,4- triazoline-3,5-dione compound.
  • Exemplary Cookson-type derivatizing agents include 4-phenyl- l,2,4-triazoline-3,5-dione (PTAD), 4-methyl-l,2,4-triazoline-3,5-dione (MTAD), 4-[2-(6,7- dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-l,2,4-triazoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)-l,2,4-triazoline-3,5- dione (NPTAD), and 4-ferrocenylmethyl- l,2,4-triazoline-3,5-dione (FMTAD). Additionally, isotopically labeled variants of Cookson-
  • the C 6 -PTAD isotopic variant is 6 mass units heavier than normal PTAD and may be used in some embodiments.
  • the C 6 -PTAD isotopic variant is 6 mass units heavier than normal PTAD and may be used in some embodiments.
  • Derivatization of steroidal compounds, including vitamin D and vitamin D related compounds, by Cookson-type reagents can be conducted by any appropriate method. See, e.g., Holmquist, et al, U.S. Patent Application Serial No. 11/946765, filed December 28, 2007;
  • mass spectrometry is performed in positive ion mode.
  • mass spectrometry is performed in negative ion mode.
  • Various ionization sources including for example atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), may be used in embodiments of the present invention.
  • APCI atmospheric pressure chemical ionization
  • ESI electrospray ionization
  • steroidal compounds including vitamin D and vitamin D related compounds, are measured using APCI in positive ion mode.
  • one or more separately detectable internal standards are provided in the sample, the amount of which are also determined in the sample.
  • all or a portion of both the analyte(s) of interest and the internal standard(s) present in the sample are ionized to produce a plurality of ions detectable in a mass spectrometer, and one or more ions produced from each are detected by mass spectrometry.
  • 2 2 internal standard(s) include vitamin D 2 -[6, 19, 19]- H 3 , vitamin D 2 -[24, 24, 24, 25, 25, 25]- 3 ⁇ 4, vitamin D 3 -[6, 19, 19]- 2 H 3 , vitamin D 3 -[24, 24, 24, 25, 25, 25]- 2 H 6 , 250HD 2 -[6, 19, 19]- 2 H 3 , 250HD 2 -[24, 24, 24, 25, 25, 25]- 2 H 6 , 250HD 3 -[6, 19, 19]- 2 H 3 , 250HD 3 -[24, 24, 24, 25, 25, 25]- 2 H 6 , la,250HD 2 -[6, 19, 19]- 2 H 3 , la,250HD 2 -[6, 19, 19]- 2 H 3 , la,250HD 2 -[24, 24, 24, 25, 25, 25]- 2 H 6 , la,250HD 3 -[6, 19, 19]- 2 H 3 , la,250HD 3 -[6, 19, 19]- 2 H 3 , la,250HD 3 -[6, 19, 19
  • One or more separately detectable internal standards may be provided in the sample prior to treatment of the sample with a Cookson-type derivatizing reagent.
  • the one or more internal standards may undergo derivatization along with the endogenous steroidal compounds, in which case ions of the derivatized internal standards are detected by mass spectrometry.
  • the presence or amount of ions generated from the analyte of interest may be related to the presence of amount of analyte of interest in the sample.
  • the internal standards may be isotopically labeled versions of steroidal compounds under investigation.
  • 250HD 2 -[6, 19, 19]- 2 H 3 or 250HD 3 -[6, 19, 19]- 2 H 3 may be used as an internal standard.
  • PTAD-250HD 2 -[6, 19, 19]- ⁇ 3 ions detectable in a mass spectrometer are selected from the group consisting of positive ions with a mass/charge ratio (m/z) of 573.30 ⁇ 0.50 and 301.10 ⁇ 0.50.
  • a PTAD-250HD 2 -[6, 19, 19]- H 3 precursor ion has a m/z of 573.30 ⁇ 0.50, and a fragment ion has m/z of 301.10 ⁇ 0.50.
  • PTAD-250HD 3 -[6, 19, 19] ions detectable in a mass spectrometer are selected from the group consisting of positive ions with a mass/charge ratio (m/z) of 561.30 ⁇ 0.50 and 301.10 ⁇ 0.50.
  • a PTAD-250HD 3 -[6, 19, 19] precursor ion has a m/z of 561.30 ⁇ 0.50, and a fragment ion has m/z of 301.10 ⁇ 0.50.
  • an "isotopic label” produces a mass shift in the labeled molecule relative to the unlabeled molecule when analyzed by mass spectrometric techniques.
  • suitable labels include deuterium ( H), C, and N.
  • 250HD 2 -[6, 19, 19] and 250HD 3 -[6, 19, 19] have masses about 3 mass units higher than 250HD 2 and 250HD 3 .
  • the isotopic label can be incorporated at one or more positions in the molecule and one or more kinds of isotopic labels can be used on the same isotopically labeled molecule.
  • the amount of the vitamin D metabolite ion or ions may be determined by comparison to one or more external reference standards.
  • Exemplary external reference standards include blank plasma or serum spiked with one or more of 250HD 2 , 250HD 2 -[6, 19, 19], 250HD 3 , and 250HD 3 -[6, 19, 19].
  • External standards typically will undergo the same treatment and analysis as any other sample to be analyzed, including treatment with one or more Cookson-type reagents prior to mass spectrometry.
  • the limit of quantitation (LOQ) of 250HD 2 is within the range of 1.9 ng/mL to 10 ng/mL, inclusive; preferably within the range of 1.9 ng/mL to 5 ng/mL, inclusive; preferably about 1.9 ng/mL.
  • the limit of quantitation (LOQ) of 25OHD 3 is within the range of 3.3 ng/mL to 10 ng/mL, inclusive; preferably within the range of 3.3 ng/mL to 5 ng/mL, inclusive; preferably about 3.3 ng/mL.
  • purification does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analytes of interest relative to other components in the sample that may interfere with detection of the analyte of interest.
  • Purification of the sample by various means may allow relative reduction of one or more interfering substances, e.g., one or more substances that may or may not interfere with the detection of selected parent or daughter ions by mass spectrometry. Relative reduction as this term is used does not require that any substance, present with the analyte of interest in the material to be purified, is entirely removed by purification.
  • solid phase extraction refers to a process in which a chemical mixture is separated into components as a result of the affinity of components dissolved or suspended in a solution (i.e., mobile phase) for a solid through or around which the solution is passed (i.e., solid phase).
  • a solution i.e., mobile phase
  • the solid phase may be retained by the solid phase resulting in a purification of the analyte in the mobile phase.
  • the analyte may be retained by the solid phase, allowing undesired components of the mobile phase to pass through or around the solid phase.
  • a second mobile phase is then used to elute the retained analyte off of the solid phase for further processing or analysis.
  • SPE including TFLC
  • Mixed mode mechanisms utilize ion exchange and hydrophobic retention in the same column; for example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention; or may exhibit column exhibit strong cation exchange and hydrophobic retention.
  • chromatography refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.
  • liquid chromatography means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s).
  • liquid chromatography include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (sometimes known as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography).
  • HPLC high performance liquid chromatography
  • HPLC high pressure liquid chromatography
  • TFLC has been applied in the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et al., J Chromatogr A 854: 23-35 (1999); see also, U.S. Patents No. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further explain TFLC.
  • laminar flow When fluid flows slowly and smoothly, the flow is called “laminar flow”. For example, fluid moving through an HPLC column at low flow rates is laminar. In laminar flow the motion of the particles of fluid is orderly with particles moving generally in straight lines. At faster velocities, the inertia of the water overcomes fluid frictional forces and turbulent flow results. Fluid not in contact with the irregular boundary "outruns” that which is slowed by friction or deflected by an uneven surface. When a fluid is flowing turbulently, it flows in eddies and whirls (or vortices), with more "drag" than when the flow is laminar.
  • Turbulent Flow Analysis Measurement and Prediction, P.S. Bernard & J.M. Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).
  • GC gas chromatography
  • large particle column or “extraction column” refers to a chromatography column containing an average particle diameter greater than about 50 ⁇ .
  • analytical column refers to a chromatography column having sufficient chromatographic plates to effect a separation of materials in a sample that elute from the column sufficient to allow a determination of the presence or amount of an analyte.
  • the analytical column contains particles of about 5 ⁇ in diameter.
  • extraction columns which have the general purpose of separating or extracting retained material from non-retained materials in order to obtain a purified sample for further analysis.
  • the terms “on-line” and “inline”, for example as used in “on-line automated fashion” or “on-line extraction” refers to a procedure performed without the need for operator intervention.
  • the term “off-line” as used herein refers to a procedure requiring manual intervention of an operator.
  • MS mass spectrometry
  • MS refers to an analytical technique to identify compounds by their mass.
  • MS refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m/z”.
  • MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means.
  • a “mass spectrometer” generally includes an ionizer and an ion detector.
  • one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometric instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”).
  • m mass
  • z charge
  • the term "operating in negative ion mode” refers to those mass spectrometry methods where negative ions are generated and detected.
  • the term "operating in positive ion mode” as used herein, refers to those mass spectrometry methods where positive ions are generated and detected.
  • the term "ionization” or “ionizing” refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.
  • EI electron ionization
  • CI chemical ionization
  • a reagent gas e.g. ammonia
  • analyte ions are formed by the interaction of reagent gas ions and analyte molecules.
  • the term "fast atom bombardment” or “FAB” refers to methods in which a beam of high energy atoms (often Xe or Ar) impacts a non-volatile sample, desorbing and ionizing molecules contained in the sample.
  • Test samples are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2- nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine.
  • a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2- nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine.
  • matrix-assisted laser desorption ionization refers to methods in which a non- volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay.
  • MALDI matrix-assisted laser desorption ionization
  • the sample is mixed with an energy- absorbing matrix, which facilitates desorption of analyte molecules.
  • the term "surface enhanced laser desorption ionization” or “SELDI” refers to another method in which a non- volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo- ionization, protonation, deprotonation, and cluster decay.
  • SELDI surface enhanced laser desorption ionization
  • the sample is typically bound to a surface that preferentially retains one or more analytes of interest.
  • this process may also employ an energy-absorbing material to facilitate ionization.
  • electrospray ionization refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.
  • APCI atmospheric pressure chemical ionization
  • mass spectrometry methods that are similar to ESI; however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure.
  • the plasma is maintained by an electric discharge between the spray capillary and a counter electrode.
  • ions are typically extracted into the mass analyzer by use of a set of differentially pumped skimmer stages.
  • a counterflow of dry and preheated N 2 gas may be used to improve removal of solvent.
  • the gas-phase ionization in APCI can be more effective than ESI for analyzing less- polar species.
  • the term "atmospheric pressure photoionization” or "APPI" as used herein refers to the form of mass spectrometry where the mechanism for the photoionization of molecule M is photon absorption and electron ejection to form the molecular ion M+. Because the photon energy typically is just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity.
  • ICP inductively coupled plasma
  • field desorption refers to methods in which a non-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.
  • LDTD Laser diode thermal desorption
  • a sample containing the analyte is thermally desorbed into the gas phase by a laser pulse.
  • the laser hits the back of a specially made 96-well plate with a metal base.
  • the laser pulse heats the base and the heat causes the sample to transfer into the gas phase.
  • the gas phase sample is then drawn into an ionization source, where the gas phase sample is ionized in preparation for analysis in the mass spectrometer.
  • ionization of the gas phase sample may be accomplished by any suitable technique known in the art, such as by ionization with a corona discharge (for example by APCI).
  • selective ion monitoring is a detection mode for a mass spectrometric instrument in which only ions within a relatively narrow mass range, typically about one mass unit, are detected.
  • multiple reaction mode sometimes known as “selected reaction monitoring” is a detection mode for a mass spectrometric instrument in which a precursor ion and one or more fragment ions are selectively detected.
  • the term "lower limit of quantification”, “lower limit of quantitation” or “LLOQ” refers to the point where measurements become quantitatively meaningful.
  • the analyte response at this LOQ is identifiable, discrete and reproducible with a relative standard deviation (RSD %) of less than 20% and an accuracy of 80% to 120%.
  • LOD limit of detection
  • an “amount" of an analyte in a body fluid sample refers generally to an absolute value reflecting the mass of the analyte detectable in volume of sample. However, an amount also contemplates a relative amount in comparison to another analyte amount. For example, an amount of an analyte in a sample can be an amount which is greater than a control or normal level of the analyte normally present in the sample.
  • Figures 1 A-D show exemplary chromatograms for PTAD-250HD 3 , PTAD-250HD 3 - [6, 19, 19]- 2 H 3 (internal standard), PTAD-250HD 2 , and PTAD-250HD 2 -[6, 19, 19]- 2 H 3 (internal standard), respectively. Details are discussed in Example 3.
  • Figures 2 A and 2B show exemplary calibration curves for 250HD 2 and 25OHD 3 in serum samples determined by methods described in Example 3.
  • Figure 3 A shows a plots of coefficient of variation versus concentration for 250HD 2 and 25OHD3.
  • Figure 3B shows the same plot expanded near the LLOQ. Details are described in Example 4.
  • Figures 4A-B show linear regression and Deming regression analyses for the comparison of mass spectrometric determination of 250HD 2 with and without PTAD
  • Figures 5 A-B show linear regression and Deming regression analyses for the comparison of mass spectrometric determination of 25OHD 3 with and without PTAD
  • Figures 6A-D show plots comparing the results of analysis of multiplex samples and unmixed samples (with the same derivatization agent). Details are described in Example 14.
  • Figures 7A-D are plots comparing the results of analysis of the same specimen treated with different derivatization agents (but comparing mixed versus mixed, or unmixed versus unmixed samples). Details are described in Example 14.
  • Figures 8A-D are plots comparing the results of analysis of the same specimen treated with different derivatization agents, with one analysis coming from a mixed sample and one coming from an unmixed sample. Details are described in Example 14.
  • Figure 9A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D 2 ions.
  • Figure 9B shows an exemplary product ion spectra (covering the m/z range of about 100 to 396) for fragmentation of the 25-hydroxyvitamin D 2 precursor ion with m/z of about 395.2. Details are described in Example 15.
  • Figure 10A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D 3 ions.
  • Figure 10B shows an exemplary product ion spectra (covering the m/z range of about 100 to 396) for fragmentation of the 25-hydroxyvitamin D 3 precursor ion with m/z of about 383.2. Details are described in Example 15.
  • Figure 11A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25-hydroxyvitamin D 2 ions.
  • Figure 1 IB shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25- hydroxyvitamin D 2 precursor ion with m/z of about 570.3. Details are described in Example 15.
  • Figure 12A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25-hydroxyvitamin D 3 ions.
  • Figure 12B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25- hydroxyvitamin D 3 precursor ion with m/z of about 558.3. Details are described in Example 15.
  • Figure 13A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l ,25-dihydroxyvitamin D 2 ions.
  • Figure 13B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- l ,25-dihydroxyvitamin D 2 precursor ion with m/z of about 550.4.
  • Figure 13C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-l ,25-dihydroxyvitamin D 2 precursor ion with m/z of about 568.4.
  • Figure 13D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-l ,25-dihydroxyvitamin D 2 precursor ion with m/z of about 586.4. Details are described in Example 16.
  • Figure 14A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l ,25-hydroxyvitamin D 3 ions.
  • Figure 14B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-l ,25- dihydroxyvitamin D 3 -PTAD precursor ion with m/z of about 538.4.
  • Figure 14C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-l ,25-dihydroxyvitamin D 3 precursor ion with m/z of about 556.4.
  • Figure 14D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-l ,25-dihydroxyvitamin D 3 precursor ion with m/z of about 574.4. Details are described in Example 16.
  • Figure 15A shows an exemplary Ql scan spectrum (covering the m/z range of about 500 to 620) for PTAD-vitamin D 2 ions.
  • Figure 15B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-vitamin D 2 precursor ion with m/z of about 572.2. Details are described in Example 17.
  • Figure 16A shows an exemplary Ql scan spectrum (covering the m/z range of about 500 to 620) for PTAD-vitamin D 3 ions.
  • Figure 16B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-vitamin D 3 precursor ion with m/z of about 560.2. Details are described in Example 17.
  • Methods are described for measuring steroidal compounds, such as vitamin D and vitamin D related compounds, in a sample. More specifically, methods are described for detecting and quantifying steroidal compounds in a plurality of test samples in a single mass spectrometric assay.
  • the methods may utilize Cookson-type reagents, such as PTAD, to generate derivatized steroidal compounds combined with methods of mass spectrometry (MS), thereby providing a high-throughput assay system for detecting and quantifying steroidal compounds in a plurality of test samples.
  • the preferred embodiments are particularly well suited for application in large clinical laboratories for automated steroidal compound quantification.
  • Suitable test samples for use in methods of the present invention include any test sample that may contain the analyte of interest.
  • a sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, etc.
  • samples are obtained from a mammalian animal, such as a dog, cat, horse, etc. Particularly preferred mammalian animals are primates, most preferably male or female humans.
  • Preferred samples comprise bodily fluids such as blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples; preferably plasma (including EDTA and heparin plasma) and serum; most preferably serum.
  • Such samples may be obtained, for example, from a patient; that is, a living person, male or female, presenting oneself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition.
  • kits for quantitation of one or more steroidal compounds may include a kit comprising the compositions provided herein.
  • a kit may include packaging material and measured amounts of an isotopically labeled internal standard, in amounts sufficient for at least one assay.
  • the kits will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged reagents for use in a steroidal compound quantitation assay.
  • Calibration and QC pools for use in embodiments of the present invention are preferably prepared using a matrix similar to the intended sample matrix.
  • one or more steroidal compounds may be enriched relative to one or more other components in the sample (e.g. protein) by various methods known in the art, including for example, liquid chromatography, filtration,
  • affinity separations including immunoaffinity separations
  • extraction methods including ethyl acetate or methanol extraction
  • chaotropic agents any combination of the above or the like.
  • Protein precipitation is one method of preparing a sample, especially a biological sample, such as serum or plasma.
  • a biological sample such as serum or plasma.
  • Protein purification methods are well known in the art, for example, Poison et ah, Journal of Chromatography B 2003, 785:263-275, describes protein precipitation techniques suitable for use in methods of the present invention.
  • test samples such as plasma or serum
  • individual test samples may be purified by a hybrid protein precipitation / liquid-liquid extraction method.
  • an unprocessed test sample is mixed with methanol, ethyl acetate, and water, and the resulting mixture is vortexed and centrifuged.
  • the resulting supernatant, containing one or more purified steroidal compounds, is removed, dried to completion and reconstituted in acetonitrile.
  • the one or more purified steroidal compounds in the acetonitrile solution may then be derivatized with any Cookson-type reagent, preferably PTAD or an isotopically labeled variant thereof.
  • LC liquid chromatography
  • Traditional HPLC analysis relies on column packing in which laminar flow of the sample through the column is the basis for separation of the analyte of interest from the sample.
  • separation in such columns is a diffusional process and may select LC, including HPLC, instruments and columns that are suitable for use with derivatized steroidal compounds.
  • the chromatographic column typically includes a medium (i.e., a packing material) to facilitate separation of chemical moieties (i.e., fractionation).
  • the medium may include minute particles, or may include a monolithic material with porous channels.
  • a surface of the medium typically includes a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties.
  • One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded, cyano bonded surface, or highly pure silica surface.
  • Alkyl bonded surfaces may include C-4, C- 8, C-12, or C-18 bonded alkyl groups.
  • the column is a highly pure silica column (such as a Thermo Hypersil Gold Aq column).
  • the chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample.
  • the sample may be supplied to the inlet port directly, or from an extraction column, such as an on-line SPE cartridge or a TFLC extraction column.
  • a multiplex sample may be purified by liquid chromatography prior to mass spectrometry.
  • the multiplex sample may be applied to the LC column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port.
  • Different solvent modes may be selected for eluting the analyte(s) of interest.
  • liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode.
  • the separation of materials is effected by variables such as choice of eluent (also known as a "mobile phase"), elution mode, gradient conditions, temperature, etc.
  • analytes may be purified by applying a multiplex sample to a column under conditions where analytes of interest are reversibly retained by the column packing material, while one or more other materials are not retained.
  • a first mobile phase condition can be employed where the analytes of interest are retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column once the non-retained materials are washed through.
  • analytes may be purified by applying a multiplex sample to a column under mobile phase conditions where the analytes of interest elute at a differential rates in comparison to one or more other materials.
  • Such procedures may enrich the amount of an analyte of interest in the eluent at a particular time (i.e, a characteristic retention time) relative to one or more other components of the sample.
  • HPLC is conducted with an alkyl bonded analytical column chromatographic system.
  • a highly pure silica column such as a Thermo Hypersil Gold Aq column
  • HPLC and/or TFLC are performed using HPLC Grade water as mobile phase A and HPLC Grade ethanol as mobile phase B.
  • valves and connector plumbing By careful selection of valves and connector plumbing, two or more chromatography columns may be connected as needed such that material is passed from one to the next without the need for any manual steps.
  • the selection of valves and plumbing is controlled by a computer pre-programmed to perform the necessary steps.
  • the chromatography system is also connected in such an on-line fashion to the detector system, e.g., an MS system.
  • the detector system e.g., an MS system.
  • an extraction column may be used for purification of steroidal compounds prior to mass spectrometry.
  • samples may be extracted using a extraction column which captures the analyte, then eluted and chromatographed on a second extraction column or on an analytical HPLC column prior to ionization.
  • sample extraction with a TFLC extraction column may be accomplished with a large particle size (50 ⁇ ) packed column.
  • Sample eluted off of this column may then be transferred to an HPLC analytical column for further purification prior to mass spectrometry. Because the steps involved in these chromatography procedures may be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. This feature may result in savings of time and costs, and eliminate the opportunity for operator error.
  • protein precipitation is accomplished with a hybrid protein precipitation / liquid-liquid extraction method which includes methanol protein precipitation and ethyl acetate/water extraction from serum test samples.
  • the resulting steroidal compounds may be derivatized prior to being subjected to an extraction column.
  • the hybrid protein precipitation / liquid-liquid extraction method and the extraction column are connected in an online fashion.
  • the extraction column is preferably a C-8 extraction column, such as a Cohesive Technologies C8XL online extraction column (50 ⁇ particle size, 0.5 x 50 mm) or equivalent.
  • the eluent from the extraction column may then be applied to an analytical LC column, such as a HPLC column in an on-line fashion, prior to mass spectrometric analysis.
  • an analytical LC column such as a HPLC column in an on-line fashion
  • derivatized steroidal compounds may be ionized by any method known to the skilled artisan. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis.
  • ionization of the sample may be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP), particle beam ionization, and LDTD.
  • ESI electron ionization
  • APCI atmospheric pressure chemical ionization
  • APPI atmospheric pressure photoionization
  • FAB fast atom bombardment
  • LSI liquid secondary ionization
  • MALDI matrix assisted laser desorption ionization
  • field ionization field desorption
  • thermospray/plasmaspray ionization
  • Derivatized steroidal compounds may be ionized in positive or negative mode.
  • derivatized steroidal compounds are ionized by APCI in positive mode.
  • derivatized steroidal compounds ions are in a gaseous state and the inert collision gas is argon or nitrogen; preferably argon.
  • mass spectrometry techniques generally, after the sample has been ionized, the positively or negatively charged ions thereby created may be analyzed to determine a mass-to- charge ratio.
  • Suitable analyzers for determining mass-to-charge ratios include quadrupole analyzers, ion traps analyzers, and time-of-flight analyzers. Exemplary ion trap methods are described in Bartolucci, et ⁇ , Rapid Commun. Mass Spectrom. 2000, 14:967-73.
  • the ions may be detected using several detection modes. For example, selected ions may be detected, i.e. using a selective ion monitoring mode (SIM), or alternatively, mass transitions resulting from collision induced dissociation or neutral loss may be monitored, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).
  • MRM multiple reaction monitoring
  • SRM selected reaction monitoring
  • the mass-to-charge ratio is determined using a quadrupole analyzer.
  • ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and the mass/charge ratio.
  • the voltage and amplitude may be selected so that only ions having a particular mass/charge ratio travel the length of the quadrupole, while all other ions are deflected.
  • quadrupole instruments may act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.
  • a precursor ion also called a parent ion
  • the precursor ion subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure.
  • fragment ions also called daughter ions or product ions
  • the MS/MS technique may provide an extremely powerful analytical tool.
  • the combination of filtration/fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples.
  • Alternate modes of operating a tandem mass spectrometric instrument include product ion scanning and precursor ion scanning.
  • product ion scanning and precursor ion scanning.
  • Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues Chapter 8 (Amadeo R. Fernandez -Alba, ed., Elsevier 2005) (387).
  • the results of an analyte assay may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, external standards may be run with the samples, and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the quantity of steroidal compounds.
  • one or more isotopically labeled vitamin D metabolites may be used as internal standards.
  • isotopically labeled vitamin D metabolites e.g., 250HD 2 -[6, 19, 19]- 2 H 3 and 250HD 3 -[6, 19, 19]- 2 H 3
  • Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.
  • One or more steps of the methods may be performed using automated machines.
  • one or more purification steps are performed on-line, and more preferably all of the purification and mass spectrometry steps may be performed in an on-line fashion.
  • precursor ions are isolated for further fragmentation though collision activated dissociation (CAD).
  • CAD collision activated dissociation
  • precursor ions gain energy through collisions with an inert gas, and subsequently fragment by a process referred to as "unimolecular decomposition.”
  • Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy.
  • Steroidal compounds in a sample may be detected and/or quantified using MS/MS as follows.
  • the samples may first be purified by protein precipitation or a hybrid protein precipitation / liquid-liquid extraction. Then, one or more steroidal compounds in the purified sample are derivatized with a Cookson-type reagent, such as PTAD or an isotopic variant thereof.
  • a Cookson-type reagent such as PTAD or an isotopic variant thereof.
  • the purified samples may then subjected to liquid chromatography, preferably on an extraction column (such as a TFLC column) followed by an analytical column (such as a HPLC column); the flow of liquid solvent from a chromatographic column enters the nebulizer interface of an MS/MS analyzer; and the solvent/analyte mixture is converted to vapor in the heated charged tubing of the interface.
  • the analyte(s) e.g., derivatized steroidal compounds such as derivatized vitamin D metabolites
  • contained in the solvent are ionized by applying a large voltage to the solvent/analyte mixture.
  • the solvent/analyte mixture nebulizes and the solvent evaporates, leaving analyte ions.
  • derivatized steroidal compounds in the purified samples may not be subject to liquid chromatography prior to ionization. Rather, the samples may be spotted in a 96-well plate and volatilized and ionized via LDTD.
  • the ions e.g. precursor ions
  • MS/MS tandem mass spectrometric
  • quadrupoles 1 and 3 are mass filters, allowing selection of ions (i.e., selection of "precursor” and “fragment” ions in Ql and Q3, respectively) based on their mass to charge ratio (m/z).
  • Quadrupole 2 is the collision cell, where ions are fragmented.
  • the first quadrupole of the mass spectrometer (Ql) selects for molecules with the mass to charge (m/z) ratios of derivatized steroidal compounds of interest.
  • Precursor ions with the correct mass/charge ratios are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass/charge ratio collide with the sides of the quadrupole and are eliminated.
  • Precursor ions entering Q2 collide with neutral argon gas molecules and fragment.
  • the fragment ions generated are passed into quadrupole 3 (Q3), where the fragment ions of derivatized steroidal compounds of interest are selected while other ions are eliminated.
  • the methods may involve MS/MS performed in either positive or negative ion mode; preferably positive ion mode. Using standard methods well known in the art, one of ordinary skill is capable of identifying one or more fragment ions of a particular precursor ion of derivatized steroidal compounds that may be used for selection in quadrupole 3 (Q3).
  • ions collide with the detector they produce a pulse of electrons that are converted to a digital signal.
  • the acquired data is relayed to a computer, which plots counts of the ions collected versus time.
  • the resulting mass chromatograms are similar to chromatograms generated in traditional HPLC-MS methods.
  • the areas under the peaks corresponding to particular ions, or the amplitude of such peaks may be measured and correlated to the amount of the analyte of interest.
  • the area under the curves, or amplitude of the peaks, for fragment ion(s) and/or precursor ions are measured to determine the amount of a particular steroidal compounds.
  • the relative abundance of a given ion may be converted into an absolute amount of the original analyte using calibration standard curves based on peaks of one or more ions of an internal molecular standard.
  • multiple patient samples can be multiplex (i.e., mixed and assayed together) if each patient sample is processed differently.
  • processed differently means that each patient sample to be included in the multiplex sample is processed in such a way that steroidal compounds in two or more patient samples that are originally indistinguishable by mass spectrometry become distinguishable after processing. This may be accomplished by processing each patient sample with a different agent that derivitizes steroidal compounds.
  • the derivatizing agents selected for use must generate derivatized steroidal compounds that are distinguishable by mass spectrometry.
  • the basis for distinguishing derivatized steroidal compounds by mass spectrometry will be a difference in the mass of ions from the derivatized steroidal compounds.
  • the differences in mass may arise from the use of two or more different derivatizing agents, such as PTAD and DMEQTAD. Differences in mass may also arise from the use of two or more isotopic variants of the same derivatizing agent, such
  • PTAD PTAD
  • C 6 -PTAD C 6 -PTAD
  • a particular steroidal compound from one patient sample will have a different mass spectrometric profile than the same steroidal compound in other patient samples.
  • processed patient samples are mixed to form a multiplex sample which is then analyzed to determine the levels of processed steroidal compounds, the differences in mass spectrometric profiles of the detected processed steroidal compounds allow for each processed steroidal compound to be attributed to an originating patient sample.
  • the amounts of a steroidal compound in two or more patient samples are determined by a single mass spectrometric analysis of a multiplex sample.
  • Cookson-type reagents may be used as derivatizing agents for different patient samples; for example, one patient sample may be derivatized with PTAD, and a second patient sample derivatized with DMEQTAD.
  • Using different Cookson-type reagents generally results in large mass differences between the derivatized analytes. For example, the difference in mass between a steroidal compound derivatized with PTAD and the same compound derivatized with DMEQTAD is about 200 mass units (the mass difference between PTAD and DMEQTAD).
  • Isotopic variants of the same Cookson-type reagent may also be used to create distinguishable derivatives in multiple patient samples.
  • one patient sample may be
  • a second patient sample may be derivatized with C 6 -PTAD.
  • the difference in mass between PTAD and C 6 -PTAD is about 6 mass units.
  • 250HD 3 -[6, 19, 19]- H 3 as internal standards. Demonstration of the methods of the present invention as applied to vitamin D metabolites does not limit the applicability of the methods to only vitamin D and vitamin D related compounds. Similarly, the use of 250HD 2 -[6, 19, 19]- H 3 or 250HD 3 -[6, 19, 19]- H 3 as internal standards are not meant to be limiting in any way. Any appropriate chemical species, easily determined by one in the art, may be used as an internal standard for steroidal compound quantitation.
  • a Perkin-Elmer Janus robot and a TomTec Quadra Tower robot was used to automate the following procedure. For each sample, 50 of serum was added to a well of a 96 well plate. Then 25 of internal standard cocktail (containing isotopically labeled 250HD 2 -[6, 19, 19]- 2 H 3 and 250HD 3 -[6, 19, 19]- 2 H 3 ) was added to each well, and the plate vortexed. Then 75 of methanol was added, followed by additional vortexing. 300 ⁇ , of ethyl acetate and 75 ⁇ , of water was then added, followed by additional vortexing, centrifugation, and transfer of the resulting supernatant to a new 96-well plate.
  • internal standard cocktail containing isotopically labeled 250HD 2 -[6, 19, 19]- 2 H 3 and 250HD 3 -[6, 19, 19]- 2 H 3
  • Sample injection was performed with a Cohesive Technologies Aria TX-4 TFLC system using Aria OS V 1.5.1 or newer software.
  • the TFLC system automatically injected an aliquot of the above prepared samples into a Cohesive Technologies C8XL online extraction column (50 ⁇ particle size, 005 x 50 mm, from Cohesive Technologies, Inc.) packed with large particles.
  • the samples were loaded at a high flow rate to create turbulence inside the extraction column. This turbulence ensured optimized binding of derivatized vitamin D metabolites to the large particles in the column and the passage of excess derivatizing reagent and debris to waste.
  • the sample was eluted off to the analytical column, a Thermo Hypersil Gold Aq analytical column (5 ⁇ particle size, 50 x 2.1 mm), with a water/ethanol elution gradient.
  • the HPLC gradient was applied to the analytical column, to separate vitamin D metabolites from other analytes contained in the sample.
  • Mobile phase A was water and mobile phase B was ethanol.
  • the HPLC gradient started with a 35 % organic gradient which was ramped to 99 % in approximately 65 seconds.
  • MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system
  • exemplary chromatograms for PTAD-250HD 3 , PTAD-250HD 3 -[6, 19, 19]- 2 H 3 (IS), PTAD-250HD 2 , and PTAD-250HD 2 -[6, 19, 19]- 2 H 3 (IS) are found in Figures 1A, IB, 1C, and ID, respectively.
  • Example 4 Analytical Sensitivity: Lower Limit of Quantitation (LLOQ) and Limit of Detection (LOP)
  • the LLOQ is the point where measurements become quantitatively meaningful.
  • the analyte response at this LLOQ is identifiable, discrete and reproducible with a precision (i.e., coefficient of variation (CV)) of greater than 20% and an accuracy of 80% to 120%.
  • the LLOQ was determined by assaying five different human serum samples spiked with PTAD-250HD 2 and PTAD-25QHD 3 at levels near the expected LLOQ and evaluating the reproducibility. Analysis of the collected data indicates that samples with concentrations of about 4 ng/mL yielded CVs of about 20%.
  • the LLOQ of this assay for both PTAD-250HD 2 and PTAD- 250HD3 was determined to be about 4 ng/mL.
  • the graphical representations of CV versus concentration for both analytes are shown in Figures 3 A-B ( Figure 3 A shows the plots over an expanded concentration range, while Figure 3B shows the same plot expanded near the LOQ).
  • the LOD is the point at which a value is beyond the uncertainty associated with its measurement and is defined as three standard deviations from the zero concentration.
  • To determine the LOD generally, blank samples of the appropriate matrix are obtained and tested for interferences. However, no appropriate biological matrix could be obtained where the endogenous concentration of 250HD 3 is zero, so a solution of 5% bovine serum albumin in phosphate buffered saline (with an estimated 1.5 ng/mL 25OHD3) was used for LOD studies. The standard was run in 20 replicates each and the resulting area rations were statistically analyzed to determine that the LOD for 250HD 2 and 25OHD 3 are about 1.9 and 3.3 ng/mL, respectively. Raw data from these studies is presented in Table 3, below
  • Intra-assay variation is defined as the reproducibility of results for a sample within a single assay.
  • QC quality control
  • Twenty replicates from each of four quality control (QC) pools covering the reportable range of the assay were prepared and measured from pooled serum with 250HD 2 and 250HD 3 at arbitrary ultralow, low, medium, and high concentrations for each analyte.
  • Acceptable levels for the coefficient of variation (CV) are less then 15% for the three higher concentration, and less than 20% for the lowest concentration (at or near the LOQ for the assay).
  • the second recovery study was performed again using six specimens. Of these six specimens, three had high endogenous concentration of 250HD 2 and three had high endogenous concentrations of 250HD 3 .
  • the specimens were paired and mixed at ratios of about 4:1, 1 : 1, and 1 :4.
  • the resulting mixtures were subjected to the hybrid protein precipitation / liquid-liquid extraction procedure described in Example 1.
  • aliquots of the extracts of the mixed specimens were derivatized with normal PTAD, following the procedure discussed above, and analyzed in quadruplicate. These experiments yielded an average accuracy of about 98% for 250HD 2 and about 93% for 250HD 3 . All individual results were within the acceptable accuracy range of 85-115%.
  • the results of the mixed specimen recovery studies are presented in Table 10, below. Table 10. Mixed Specimen Recovery Studies
  • ⁇ Measured values are averages of analysis of four aliquots.
  • 2006/0228808 (Caulfield, et al). Eight specimens were split and analyzed according to both methods. The correlation between the two methods was assessed with linear regression, deming regression, and Bland-Altman analysis for complete data sets (including calibration samples, QC pools, and unknowns), as well as for unknowns only.
  • Hemolysis The effect of hemolysis was evaluated by pooling patient samples with known endogenous concentrations of both 250HD 2 and 250HD 3 to create five different pools with concentrations across the dynamic range of the assay. Then, lysed whole blood was spiked into the pools to generate lightly and moderately hemolyzed samples.
  • Lipemia The effect of lipemia was evaluated by pooling patient samples with known endogenous concentrations of both 250HD 2 and 250HD 3 to create five different pools with concentrations across the dynamic range of the assay. Then, powdered lipid extract was added to the pools to generate lightly and grossly lipemic specimens. Specimens were run in
  • Icteria The effect of icteria was evaluated by pooling patient samples with known endogenous concentrations of both 250HD 2 and 250HD 3 to create five different pools with concentrations across the dynamic range of the assay. Then, a concentrated solution of Bilirubin was spiked into the pools to generate lightly and grossly icteric specimens. Specimens were run in quadruplicate and results were compared to the non-icteric pool result and the accuracy was calculated. The data showed that 250HD 2 and 250HD 3 are unaffected by icteria (with all values within an acceptable accuracy range of 85-115%). Therefore, icteric specimens are acceptable.
  • Example 12 Injector Carryover Studies
  • the assay was conducted on various specimen types. Human serum and Gel-Barrier Serum (i.e., serum from Serum Separator Tubes), as well as EDTA Plasma and Heparin were established as acceptable sample types. In these studies, sets of human serum (serum), Gel- Barrier Serum (SST), EDTA Plasma (EDTA), and heparin (Na Hep) drawn at the same time from the same patient were tested for 250HD 2 (40 specimen sets) and 25OHD 3 (6 specimen sets). Due to the limitations with clot detection/sensing in existing automated pipetting systems, plasma was not tested for automated procedures.
  • Example 14 Multiplex Patient Samples with Multiple Derivatizing Agents
  • sample A and sample B Two patients samples (i.e., sample A and sample B) were both subjected to the hybrid protein precipitation / liquid-liquid extraction procedure described in Example 1. Then, aliquots of the extracts from sample A and sample B were derivatized with normal PTAD, following the procedure discussed above. Second aliquots of the extracts from sample A and sample B were derivatized with normal PTAD, following the procedure discussed above. Second aliquots of the extracts from sample A and
  • sample B were also derivatized with C 6 -PTAD, also according to the procedure discussed above.
  • Figures 6A-D are plots comparing the results of analysis of multiplex samples and unmixed samples (with the same derivatization agent). These plots show R values in excellent agreement (i.e., R values for all four variants are in excess of 0.98). This shows that, given a constant derivatization agent, analysis of mixed samples gives the same result as analysis of unmixed samples.
  • Figures 7A-D are plots comparing the results of analysis of the same specimen treated with different derivatization agents (but comparing mixed versus mixed, or unmixed versus
  • C 6 -PTAD is not a source of difference in the performance of the assay, at least when the compared samples are both mixed, or unmixed.
  • Figures 8A-D are plots comparing the results of analysis of the same specimen treated with different derivatization agents, with one analysis coming from a mixed sample and one coming from an unmixed sample. These plots also show R values in excellent agreement (i.e., R values for all four variants are in excess of 0.99). This shows that the isotopic variation between PTAD and C 6 -P AD in combination with variation between mixed and unmixed samples is not a source of difference in the performance of the assay.
  • isotopic variation of the PTAD derivatization agent made no meaningful difference even when samples were mixed together and introduced into the mass spectrometer as a single injection. Multiplexing of patient samples was successfully demonstrated.
  • Example 15 Exemplary spectra from LDTD-MS/MS analysis of native and PTAD derivatized 25-hydroxyvitamin D? and 25-hydroxyvitamin D 3
  • Exemplary Ql scan spectra from analysis of 25-hydroxyvitamin D 2 and 25- hydroxyvitamin D 3 are shown in Figures 9A and 10A, respectively. These spectra were collected by scanning Ql across a m/z range of about 350 to 450.
  • a preferred MRM transition for the quantitation of 25-hydroxyvitamin D 2 is fragmenting a precursor ion with a m/z of about 395.2 to a product ion with a m/z of about 208.8 or 251.0.
  • a preferred MRM transition for the quantitation of 25-hydroxyvitamin D 3 is fragmenting a precursor ion with a m/z of about 383.2 to a product ion with a m/z of about 186.9 or 257.0.
  • additional product ions may be selected to replace or augment the preferred fragment ion.
  • Exemplary Ql scan spectra from the analysis of samples containing PTAD-25- hydroxyvitamin D 2 and PTAD-25-hydroxyvitamin D3 are shown in Figures 11 A and 12 A, respectively. These spectra were collected by scanning Ql across a m/z range of about 520 to 620.
  • a preferred MRM transition for the quantitation of PTAD-25-hydroxyvitamin D 2 is fragmenting a precursor ion with a m/z of about 570.3 to a product ion with a m/z of about 298.1.
  • a preferred MRM transition for the quantitation of PTAD-25-hydroxyvitamin D3 is fragmenting a precursor ion with a m/z of about 558.3 to a product ion with a m/z of about 298.1.
  • additional product ions may be selected to replace or augment the preferred fragment ion.
  • Example 16 Exemplary spectra from LDTD-MS/MS analysis of PTAD derivatized la,25- dihydroxyvitamin D? and l ,25-dihydroxyvitamin D ⁇
  • PTAD derivatives of l ,25-dihydroxyvitamin D 2 and l ,25-dihydroxyvitamin D3 were prepared by treating aliquots of stock solutions of each analyte with PTAD in acetonitrile. The derivatization reactions was allowed to proceed for approximately one hour, and were quenched by adding water to the reaction mixture. The derivatized analytes were then analyzed according to the LDTD-MS/MS procedure outlined above.
  • Exemplary Ql scan spectra from the analysis of samples containing PTAD-la,25- dihydroxyvitamin D 2 and PTAD-l ,25-hydroxyvitamin D3 are shown in Figures 13 A, and 14 A, respectively. These spectra were collected with LDTD-MS/MS by scanning Ql across a m/z range of about 520 to 620.
  • Exemplary MRM transitions for the quantitation of PT AD- 1 a,25 -dihydroxyvitamin D 2 include fragmenting a precursor ion with a m/z of about 550.4 to a product ion with a m/z of about 277.9; fragmenting a precursor ion with a m/z of about 568.4 to a product ion with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of about 586.4 to a product ion with a m/z of about 314.2.
  • Exemplary MRM transitions for the quantitation of PTAD-la,25- hydroxyvitamin D 3 include fragmenting a precursor ion with a m/z of about 538.4 to a product ion with a m/z of about 278.1 ; fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m z of about 298.0; and fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 313.0.
  • the product ion scans in Figures 6B-D and 7B-D several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 13B-D and 14B-D to replace or augment the exemplary fragment ions.
  • PTAD derivatives of various deuterated forms of dihydroxyvitamin D metabolites were also investigated.
  • Exemplary MRM transitions for the quantitation of PTAD- 1 a,25 -dihydroxyvitamin D 2 -[26, 26, 27, 27, 27]- 3 ⁇ 4 include fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m/z of about 278.1 ; fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 298.1; and fragmenting a precursor ion with a m/z of about 592.4 to a product ion with a m/z of about 313.9.
  • Exemplary MRM transitions for the quantitation of PTAD- 1 a,25 -dihydroxyvitamin D -[6, 19, 19]- H include fragmenting a precursor ion with a m/z of about 541.4 to a product ion with a m/z of about 280.9; fragmenting a precursor ion with a m/z of about 559.4 to a product ion with a m/z of about 301.1; and fragmenting a precursor ion with a m/z of about 577.4 to a product ion with a m/z of about 317.3.
  • Exemplary MRM transitions for the quantitation of PTAD- la,25 -dihydroxyvitamin D 2 -[26, 26, 27, 27, 27]- H 6 include fragmenting a precursor ion with a m/z of about 544.4 to a product ion with a m/z of about 278.0; fragmenting a precursor ion with a m/z of about 562.4 to a product ion with a m/z of about 298.2; and fragmenting a precursor ion with a m/z of about 580.4 to a product ion with a m/z of about 314.0.
  • Example 17 Exemplary spectra from MS/MS analysis of PTAD derivatized vitamin D? and vitamin D
  • PTAD derivatives of vitamin D 2 , and vitamin D 3 were prepared by treating aliquots of stock solutions of each analyte with PTAD in acetonitrile. The derivatization reactions was allowed to proceed for approximately one hour, and were quenched by adding water to the reaction mixture. The derivatized analytes were then analyzed by MS/MS.
  • Exemplary Ql scan spectra from the analysis of samples containing PTAD-vitamin D 2 , and PTAD-vitamin D 3 are shown in Figures 15A and 16A, respectively. These analyses were conducted by directly injecting standard solutions containing the analyte of interest into a Finnigan TSQ Quantum Ultra MS/MS system (Thermo Electron Corporation). A liquid chromatography mobile phase was simulated by passing 800 ⁇ / ⁇ of 80% acetonitrile, 20%> water with 0.1% formic acid through an HPLC column, upstream of introduction of the analyte. The spectra were collected by scanning Ql across a m/z range of about 500 to 620.
  • Exemplary product ion scans generated from precursor ions for each of PTAD- vitamin D 2 and PTAD-vitamin D3 are presented in Figures 15B and 16B, respectively.
  • the precursor ions selected in Q 1 and the collision energies used to generate these product ion spectra are indicated in Table 17.
  • An exemplary MRM transition for the quantitation of PTAD-vitamin D 2 includes fragmenting a precursor ion with a m/z of about 572.2 to a product ion with a m/z of about 297.9.
  • An exemplary MRM transition for the quantitation of PTAD-vitamin D3 includes fragmenting a precursor ion with a m/z of about 560.2 to a product ion with a m/z of about 298.0.
  • several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 15B and 16B to replace or augment the exemplary fragment ions.
  • PTAD derivatives of various deuterated forms of vitamin D were also investigated.
  • PTAD derivatives of vitamin D 2 -[6, 19, 19]- 2 H 3 vitamin D 2 -[26, 26, 26, 27, 27, 27]- 2 H 6 ,
  • vitamin D 3 -[6, 19, 19]- H 3 and vitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 were prepared and analyzed as above.
  • An exemplary MRM transition for the quantitation of PTAD-vitamin D 2 -[6, 19, 19]- H 3 includes fragmenting a precursor ion with a m/z of about 575.2 to a product ion with a m/z of about 301.0.
  • An exemplary MRM transition for the quantitation of PTAD-vitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 includes fragmenting a precursor ion with a m z of about 578.2 to a product ion with a m/z of about 297.9.
  • An exemplary MRM transition for the quantitation of PTAD-vitamin D 3 -[6, 19, 19]- H 3 includes fragmenting a precursor ion with a m/z of about 563.2 to a product ion with a m/z of about 301.0.
  • An exemplary MRM transition for the quantitation of PTAD-vitamin ⁇ -[26, 26, 26, 27, 27, 27]- H 6 includes fragmenting a precursor ion with a m/z of about 566.2 to a product ion with a m/z of about 298.0.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Optics & Photonics (AREA)
  • Endocrinology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
PCT/US2010/059746 2009-12-11 2010-12-09 Mass spectrometry of steroidal compounds in multiplex samples WO2011072152A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
EP19182389.7A EP3693734A3 (en) 2009-12-11 2010-12-09 Mass spectrometry of steroidal compounds in multiplex samples
CN201080063451.XA CN102812356B (zh) 2009-12-11 2010-12-09 多重样品中的甾族化合物的质谱法
CA2783708A CA2783708C (en) 2009-12-11 2010-12-09 Mass spectrometry of steroidal compounds in multiplex samples
US13/514,892 US9034653B2 (en) 2009-12-11 2010-12-09 Mass spectrometry of steroidal compounds in multiplexed patient samples
JP2012543287A JP5739903B2 (ja) 2009-12-11 2010-12-09 複合試料中のステロイド化合物の質量分析
EP10836702.0A EP2510347B1 (en) 2009-12-11 2010-12-09 Mass spectrometry of steroidal compounds in multiplex samples
US14/715,153 US9506937B2 (en) 2009-12-11 2015-05-18 Mass spectrometry of steroidal compounds in multiplexed patient samples
US15/362,210 US10955424B2 (en) 2009-12-11 2016-11-28 Mass spectrometry of steroidal compounds in multiplexed patient samples
US17/208,564 US11808773B2 (en) 2009-12-11 2021-03-22 Mass spectrometry of steroidal compounds in multiplexed patient samples
US18/364,356 US20230375575A1 (en) 2009-12-11 2023-08-02 Mass spectrometry of steroidal compounds in multiplex samples

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US28594109P 2009-12-11 2009-12-11
US61/285,941 2009-12-11

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US13/514,892 A-371-Of-International US9034653B2 (en) 2009-12-11 2010-12-09 Mass spectrometry of steroidal compounds in multiplexed patient samples
US14/715,153 Continuation US9506937B2 (en) 2009-12-11 2015-05-18 Mass spectrometry of steroidal compounds in multiplexed patient samples

Publications (1)

Publication Number Publication Date
WO2011072152A1 true WO2011072152A1 (en) 2011-06-16

Family

ID=44145921

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/059746 WO2011072152A1 (en) 2009-12-11 2010-12-09 Mass spectrometry of steroidal compounds in multiplex samples

Country Status (6)

Country Link
US (5) US9034653B2 (ja)
EP (2) EP3693734A3 (ja)
JP (5) JP5739903B2 (ja)
CN (2) CN107607662B (ja)
CA (2) CA3013468C (ja)
WO (1) WO2011072152A1 (ja)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014152502A1 (en) * 2013-03-15 2014-09-25 Novilytic, LLC Method for determining derivatized analytes in a separated biological fluid
JP2015508504A (ja) * 2012-01-20 2015-03-19 ディーエイチ テクノロジーズ デベロップメント プライベート リミテッド 標識化学およびｌｃ−ｍｓｍｓを使用するエストラジオールおよびフェノール性ｏｈを有する分析物の分析
EP3301453A1 (en) * 2016-09-29 2018-04-04 Tokyo University of Science Method for derivatizing an s-cis-diene compound, derivatization reagent kit, and method for analyzing an s-cis-diene compound
CN111936851A (zh) * 2018-01-29 2020-11-13 Dh科技发展私人贸易有限公司 用于检测维生素d代谢物的方法和系统
US10935558B2 (en) 2005-04-06 2021-03-02 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites by mass spectrometry
US10955424B2 (en) 2009-12-11 2021-03-23 Quest Diagnostics Investments Incorporated Mass spectrometry of steroidal compounds in multiplexed patient samples
US11105821B2 (en) 2009-12-03 2021-08-31 Quest Diagnostics Investments Incorporated Vitamin D metabolite determination utilizing mass spectrometry following derivatization
US11193948B2 (en) 2016-11-17 2021-12-07 Tokyo University Of Science Reagent kit and method for derivatizing and quantifying vitamin D using a mass spectrometer
US11280799B2 (en) 2009-12-11 2022-03-22 Quest Diagnostics Investments Incorporated Mass spectrometric determination of non-derivatized, non-metabolized vitamin D
US11650216B2 (en) 2007-11-28 2023-05-16 Quest Diagnostics Investments Incorporated Methods for detecting dihydroxyvitamin D metabolites by mass spectrometry

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9024039B2 (en) * 2005-12-12 2015-05-05 Women & Infants' Hospital Of Rhode Island Heterocycles and derivatives thereof and methods of manufacture and therapeutic use
CN103743839A (zh) * 2013-12-30 2014-04-23 西北工业大学 测定正辛基二茂铁及杂质含量的hplc方法
CN107002017B (zh) * 2014-12-17 2020-07-28 西门子医疗保健诊断公司 疏水性半抗原分析物的精确测定测量
KR101984623B1 (ko) * 2017-05-12 2019-05-31 강원대학교산학협력단 트리아졸디온 기반 화합물을 이용한 비타민 d결합 및 형광이 담지된 실리카 나노입자를 이용한 비타민 d의 정량 검출 방법
MX2020009617A (es) 2018-03-16 2020-12-07 Quest Diagnostics Invest Llc Determinación espectrométrica de masa de testosterona en muestra multplexada de un paciente.
CN112218868B (zh) * 2018-06-12 2023-12-08 株式会社德山 三唑啉二酮加成物及其制造方法、烯化合物的制造方法及分析方法
EP3712137A4 (en) 2018-06-12 2021-08-18 Tokuyama Corporation METHOD FOR MAKING A SOLID TRIAZOLININDIONE COMPOUND, SOLID TRIAZOLINONE COMPOUND AND METHOD FOR MAKING A TRIAZOLININDIONE COMPOUND
CN109187827B (zh) * 2018-08-14 2021-06-18 成都益康谱科技有限公司 人体尿液中雌激素及其代谢产物在线检测方法
US10803981B2 (en) 2018-10-15 2020-10-13 James Glenn Norman Applied artificial intelligence technology for hormone therapy treatment
US11885819B2 (en) 2019-10-09 2024-01-30 Joel Ltd Analysis method of diene compound
US11906510B2 (en) * 2020-03-20 2024-02-20 Stanford Junior University Metabolic profiling by reverse-phase/ion-exchange mass spectrometry
WO2022049726A1 (ja) * 2020-09-04 2022-03-10 株式会社アデランス 胆汁酸類、ステロール類、及びホルモン類の分析方法及び分析システム
JP6806948B1 (ja) * 2020-09-04 2021-01-06 株式会社アデランス 胆汁酸類、ステロール類、及びホルモン類の分析方法及び分析システム

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060094125A1 (en) * 2004-10-29 2006-05-04 Singh Ravinder J Vitamin D deficiencies
US20080241955A1 (en) * 2006-06-30 2008-10-02 Applera Corporation Methods, Mixtures, Kits And Compositions Pertaining To Analyte Determination
US20090137056A1 (en) * 2007-11-28 2009-05-28 Brett Holmquist Methods for detecting dihydroxyvitamin d metabolites by mass spectrometry

Family Cites Families (65)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4332591A (en) 1978-01-19 1982-06-01 Sumitomo Chemical Company, Limited Analytical method and apparatus for the determination of total nitrogen contents in aqueous systems
US5030772A (en) 1990-02-14 1991-07-09 Deluca Hector F Process for preparing vitamin D2 compounds and the corresponding 1 α-hydroxylated derivatives
US5605798A (en) 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
JPH06341980A (ja) 1993-02-24 1994-12-13 Kureha Chem Ind Co Ltd ビタミンｄ３誘導体の定量法
NZ267842A (en) 1993-05-28 1997-09-22 Baylor College Medicine Apparatus for measuring molecular mass (by mass spectrometry) in which the sample is ionised on the sample holder and desorbed therefrom by laser pulses
JP2968910B2 (ja) 1993-07-14 1999-11-02 ヤマサ醤油株式会社 １α，２５（ＯＨ）２ビタミンＤ３に対する抗体及びその用途
WO1995033279A1 (en) 1994-05-31 1995-12-07 University Of Warwick Tandem mass spectrometry apparatus
DE19531152A1 (de) 1994-12-15 1996-06-20 Bayer Ag Substituierte N-Aryl-Stickstoffheterocyclen
US5614408A (en) 1995-10-18 1997-03-25 The United States Of America As Represented By The Secretary Of Agricultural Monoclonal antibodies to potato, tomato, and eggplant glycoalkaloids and assays for the same
US5772874A (en) 1995-11-02 1998-06-30 Cohesive Technologies, Inc. High performance liquid chromatography method and apparatus
PL327920A1 (en) 1996-01-19 1999-01-04 Cohesive Technologies Highly efficient liquid chromatography process and chromatographic apparatus therefor
CA2270132A1 (en) 1996-11-06 1998-05-14 Sequenom, Inc. Dna diagnostics based on mass spectrometry
US5916523A (en) 1996-12-04 1999-06-29 Antek Instruments, Inc. Methods for near simultaneous chemiluminescent sulfur and nitrogen detection
US20010007907A1 (en) 1997-05-16 2001-07-12 Women And Infants Hospital 3-epi compounds of vitamin d3 and uses thereof
GB9717926D0 (en) 1997-08-22 1997-10-29 Micromass Ltd Methods and apparatus for tandem mass spectrometry
GB9719187D0 (en) 1997-09-09 1997-11-12 Glaxo Group Ltd Compounds
US6040575A (en) 1998-01-23 2000-03-21 Analytica Of Branford, Inc. Mass spectrometry from surfaces
DE19981132D2 (de) 1998-06-25 2001-10-18 Immundiagnostik Ges Fuer Produ Funktionelle Vitamin-D-Derivate und Verfahren zur Bestimmung von 25-Hydroxy- und 1alpha,25-Dihydroxy-Vitamin-D
US20020045606A1 (en) 1999-12-02 2002-04-18 Reddy Satyanarayana G. Esters of vitamin D3 and uses thereof
GB0029729D0 (en) 2000-12-06 2001-01-17 Ids Ltd Method for detection of vitamin D metabolites
US7087395B1 (en) 2001-01-16 2006-08-08 Quest Diagnostics Investments Incorporated Vitamin D assay
EP1461447A4 (en) 2001-11-29 2006-08-23 Thermo Finnigan Llc QUANTIFICATION OF POLYPEPTIDES
TWI286554B (en) 2002-06-27 2007-09-11 Chugai Pharmaceutical Co Ltd Ferrocene compounds and uses thereof
US7632686B2 (en) 2002-10-03 2009-12-15 Anderson Forschung Group High sensitivity quantitation of peptides by mass spectrometry
CA2501000C (en) 2002-10-03 2015-05-26 Norman Leigh Anderson High sensitivity quantitation of peptides by mass spectrometry
US7618827B2 (en) 2003-04-14 2009-11-17 Georgetown University Thyroid hormone analysis by mass spectrometry
WO2004090525A1 (en) 2003-04-14 2004-10-21 Georgetown University Steroid hormone analysis by mass spectrometry
US6977143B1 (en) 2003-09-08 2005-12-20 Quest Diagnostics Investments Incorporated Determination of testosterone by mass spectrometry
EP1687643A4 (en) * 2003-11-14 2010-11-24 Irm Llc FLUORESCENT MARKING FOR SELECTIVE TREATMENT OF BIOLOGICALLY DERIVED SAMPLES
EP1717512A4 (en) 2003-12-19 2007-03-07 Hitachi Chemical Co Ltd BACKLIGHT DEVICE AND LIQUID CRYSTAL DISPLAY
WO2005117906A1 (en) * 2004-05-19 2005-12-15 Merck & Co., Inc. Combination for inhibiting bone resorption comprising a bisphosponate (alendrona) and a vitamin d (cholecalciferol)
CA2480549A1 (fr) 2004-09-15 2006-03-15 Phytronix Technologies Inc. Source d'ionisation pour spectrometre de masse
JP2008513031A (ja) 2004-09-20 2008-05-01 プロテオジェニックス， インコーポレイテッド 胎児異数性の診断
US20070037286A1 (en) 2005-02-09 2007-02-15 Subhasish Purkayastha Thyroxine-containing compound analysis methods
WO2006107339A2 (en) 2005-03-31 2006-10-12 Georgetown University Free thyroxine and free triiodothyronine analysis by mass spectrometry
US7972867B2 (en) 2005-04-06 2011-07-05 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites by mass spectrometry
US7745226B2 (en) 2005-04-06 2010-06-29 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites
US7892845B2 (en) 2005-04-06 2011-02-22 Washington University In St. Louis Methods for measuring the metabolism of neurally dervied biomolecules in vivo
US20060236886A1 (en) 2005-04-20 2006-10-26 Agfa-Gevaert Process for the offset printing of a catalytic species via a hydrophilic phase
EP1931711B1 (en) 2005-09-29 2009-04-08 Roche Diagnostics GmbH Antibodies against 25-hydroxyvitamin d
US20070105179A1 (en) 2005-11-09 2007-05-10 Dionex Corporation Methods of detecting N-and O-linked oligosaccharides in glycoproteins
US7855057B2 (en) 2006-03-23 2010-12-21 Millipore Corporation Protein splice variant/isoform discrimination and quantitative measurements thereof
US7989760B2 (en) 2006-05-24 2011-08-02 SWCE Inc. Extraction detection system and method
CA2820957A1 (en) 2006-05-26 2007-12-06 Laboratory Corporation Of America Holdings Liquid chromatography with tandem mass spectrometry of estrone, estradiol and free thyroxine
CN101467048B (zh) 2006-06-06 2014-04-02 霍夫曼-拉罗奇有限公司 改良的维生素d测量方法
US20100288029A1 (en) 2006-08-30 2010-11-18 Ge Healthcar Bio-Sciences Corp. System and method for characterizing membranes and membrane filtration devices
US7999084B2 (en) 2007-05-16 2011-08-16 Agilent Technologies, Inc. Devices and methods for reducing matrix effects
US7807172B2 (en) 2007-06-13 2010-10-05 University Of Washington Methods and compositions for detecting thyroglobulin in a biological sample
WO2008156139A1 (ja) 2007-06-21 2008-12-24 National University Corporation University Of Fukui 同位体化合物を標識として使用するタンパク質の分析方法
JP5080945B2 (ja) * 2007-11-09 2012-11-21 株式会社日立ハイテクノロジーズ 質量分析装置および質量分析方法
US8030084B2 (en) 2007-12-06 2011-10-04 Quest Diagnostics Investments Incorporated Thyroglobulin quantitation by mass spectrometry
US20090317916A1 (en) * 2008-06-23 2009-12-24 Ewing Kenneth J Chemical sample collection and detection device using atmospheric pressure ionization
WO2010019566A2 (en) 2008-08-11 2010-02-18 Mayo Foundation For Medical Education And Research Methods for determining levels of 1,25 dihydroxy vitamin d2 and d3
WO2010099108A2 (en) * 2009-02-24 2010-09-02 Mayo Foundation For Medical Education And Research Multiplexing derivatized anayltes using mass spectroscopy
US8034627B2 (en) 2009-12-03 2011-10-11 Quest Diagnostics Investments Incorporated Methods for detecting dihydroxyvitamin D metabolites by mass spectrometry
US7977117B2 (en) 2009-12-03 2011-07-12 Quest Diagnostics Investments Incorprated Vitamin D metabolite determination utilizing mass spectrometry following derivatization
US9034653B2 (en) 2009-12-11 2015-05-19 Quest Diagnostics Investments Inc. Mass spectrometry of steroidal compounds in multiplexed patient samples
US20120025067A1 (en) 2009-12-11 2012-02-02 Quest Diagnostics Investments Incorporated Mass spectrometric determination of non-derivatized, non-metabolized vitamin d
CA2787900C (en) 2010-01-25 2018-10-02 Dh Technologies Development Pte. Ltd. Quantitative analysis of vitamin d3, vitamin d2, and metabolites thereof
US9274124B2 (en) 2010-03-15 2016-03-01 Anderson Forschung Group, Inc. Mass spectrometric assays for peptides
EP2633325B1 (en) 2010-10-29 2016-12-28 Cohesive Technologies Inc. Lc-ms separation and detection of vitamin d metabolites
WO2012111249A1 (ja) 2011-02-14 2012-08-23 学校法人麻布獣医学園 質量分析法における質量変化を検出する方法及び安定同位体標識タンパク質の絶対量の定量方法
US9140695B2 (en) 2012-01-18 2015-09-22 University Of Utah Research Foundation Methods for analysis of free and autoantibody-bound biomarkers and associated compositions, devices, and systems
CN104797715A (zh) 2012-09-20 2015-07-22 奎斯特诊断投资公司 通过质谱法定量甲状腺球蛋白
US10116605B2 (en) 2015-06-22 2018-10-30 Cisco Technology, Inc. Transport stack name scheme and identity management

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060094125A1 (en) * 2004-10-29 2006-05-04 Singh Ravinder J Vitamin D deficiencies
US20080241955A1 (en) * 2006-06-30 2008-10-02 Applera Corporation Methods, Mixtures, Kits And Compositions Pertaining To Analyte Determination
US20090137056A1 (en) * 2007-11-28 2009-05-28 Brett Holmquist Methods for detecting dihydroxyvitamin d metabolites by mass spectrometry

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10935558B2 (en) 2005-04-06 2021-03-02 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites by mass spectrometry
US11921122B2 (en) 2005-04-06 2024-03-05 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites by mass spectrometry
US11579154B2 (en) 2005-04-06 2023-02-14 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites by mass spectrometry
US11650216B2 (en) 2007-11-28 2023-05-16 Quest Diagnostics Investments Incorporated Methods for detecting dihydroxyvitamin D metabolites by mass spectrometry
US11885820B2 (en) 2009-12-03 2024-01-30 Quest Diagnostics Investments Incorporated Vitamin D metabolite determination utilizing mass spectrometry following derivatization
US11105821B2 (en) 2009-12-03 2021-08-31 Quest Diagnostics Investments Incorporated Vitamin D metabolite determination utilizing mass spectrometry following derivatization
US11549954B2 (en) 2009-12-11 2023-01-10 Quest Diagnostics Investments Incorporated Mass spectrometric determination of non-derivatized, non-metabolized vitamin D
US10955424B2 (en) 2009-12-11 2021-03-23 Quest Diagnostics Investments Incorporated Mass spectrometry of steroidal compounds in multiplexed patient samples
US11280799B2 (en) 2009-12-11 2022-03-22 Quest Diagnostics Investments Incorporated Mass spectrometric determination of non-derivatized, non-metabolized vitamin D
US11808773B2 (en) 2009-12-11 2023-11-07 Quest Diagnostics Investments Incorporated Mass spectrometry of steroidal compounds in multiplexed patient samples
US11852636B2 (en) 2009-12-11 2023-12-26 Quest Diagnostics Investments Incorporated Mass spectrometric determination of non-derivatized, non-metabolized vitamin D
JP2015508504A (ja) * 2012-01-20 2015-03-19 ディーエイチ テクノロジーズ デベロップメント プライベート リミテッド 標識化学およびｌｃ−ｍｓｍｓを使用するエストラジオールおよびフェノール性ｏｈを有する分析物の分析
WO2014152502A1 (en) * 2013-03-15 2014-09-25 Novilytic, LLC Method for determining derivatized analytes in a separated biological fluid
US9068995B2 (en) 2013-03-15 2015-06-30 Novilytic, LLC Method for determining derivatized analytes in a separated biological fluid
US10761102B2 (en) 2016-09-29 2020-09-01 Tokyo University Of Science Method for derivatizing an s-cis-diene compound, derivatization reagent kit, and method for analyzing an s-cis-diene compound
EP3301453A1 (en) * 2016-09-29 2018-04-04 Tokyo University of Science Method for derivatizing an s-cis-diene compound, derivatization reagent kit, and method for analyzing an s-cis-diene compound
US11193948B2 (en) 2016-11-17 2021-12-07 Tokyo University Of Science Reagent kit and method for derivatizing and quantifying vitamin D using a mass spectrometer
CN111936851A (zh) * 2018-01-29 2020-11-13 Dh科技发展私人贸易有限公司 用于检测维生素d代谢物的方法和系统
EP3746781A4 (en) * 2018-01-29 2021-11-24 DH Technologies Development Pte. Ltd. VITAMIN D METABOLITE DETECTION METHODS AND SYSTEMS

Also Published As

Publication number Publication date
CA2783708C (en) 2018-09-18
JP2017116551A (ja) 2017-06-29
US20170261523A1 (en) 2017-09-14
JP2018077250A (ja) 2018-05-17
JP6585743B2 (ja) 2019-10-02
JP5739903B2 (ja) 2015-06-24
CN102812356B (zh) 2017-08-08
JP2020012837A (ja) 2020-01-23
JP6276435B2 (ja) 2018-02-07
CN102812356A (zh) 2012-12-05
US20160061848A1 (en) 2016-03-03
US10955424B2 (en) 2021-03-23
EP2510347A4 (en) 2013-10-30
CA3013468C (en) 2020-11-10
EP3693734A3 (en) 2020-12-23
EP2510347A1 (en) 2012-10-17
JP6092289B2 (ja) 2017-03-08
US20210349109A1 (en) 2021-11-11
US20230375575A1 (en) 2023-11-23
JP2015166740A (ja) 2015-09-24
JP2013513800A (ja) 2013-04-22
EP3693734A2 (en) 2020-08-12
JP6923603B2 (ja) 2021-08-25
US11808773B2 (en) 2023-11-07
US9506937B2 (en) 2016-11-29
CA3013468A1 (en) 2011-06-16
US20130137185A1 (en) 2013-05-30
CN107607662B (zh) 2020-05-08
CA2783708A1 (en) 2011-06-16
EP2510347B1 (en) 2019-03-20
US9034653B2 (en) 2015-05-19
CN107607662A (zh) 2018-01-19

Similar Documents

Publication Publication Date Title
US11808773B2 (en) Mass spectrometry of steroidal compounds in multiplexed patient samples
US11885820B2 (en) Vitamin D metabolite determination utilizing mass spectrometry following derivatization
US11549954B2 (en) Mass spectrometric determination of non-derivatized, non-metabolized vitamin D

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201080063451.X

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10836702

Country of ref document: EP

Kind code of ref document: A1

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10836702

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2783708

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2012543287

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 6090/DELNP/2012

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: 2010836702

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 13514892

Country of ref document: US